Proteomics based approach to understanding tissue regeneration Seastar as a model organism -Preliminary results- Catarina Franco1, Romana Santos1, Ana Varela Coelho1,2 1. Instituto de Tecnologia Química e Biológica, Oeiras, Portugal; 2. Universidade de Évora, Évora, Portugal; Therefore, echinoderms are potencial animal models to understand which biomolecules (proteins and peptides) are involved in these processes. Preliminary experiments have enabled the collection of coelomic fluid, which is in contact with all the organs and may thus act as a carrier of molecules and proteins important for the process of regeneration. Preparation of the coelomic fluid was optimized in order to analyse insoluble proteins by two-dimensional electrophoresis (2DE) and soluble proteins and neuropeptides by LC-MS/MS. Marthasterias glacialis (Linné, 1758), a common sea star of the Portuguese coast, is being used as a model system to identify proteins and neuropeptides that are implicated in the regeneration process. Regeneration is a common trait in all echinoderm classes being used to replace external and internal organs, which are lost following traumatic injury, predation, autotomy or as a reproduction strategy. Fig.1. Seastar regenerating two arms. 2 Coelomic fluid collection Seastars were kept in re-circulating aquariums at the Vasco da Gama Aquarium (Dafundo, Oeiras). Fig.2. Seastars in the re-circulating aquariums (Vasco da Gama Aquarium). Fig.3. Seastar under surgery. Coelomic fluid localization. (B) Coelomic fluid collection (A) (B) The coelomic fluid is in contact with all internal organs of the seastar. 1 Coelomic fluid fractionation Antiprotease mixture (Sigma) was added before any sample handeling Several centrifugation speeds were tested in order to separe the material in two different fractions: Insoluble: all that precipitate with centrifugation; Soluble: all that stays in the centrifugation supernatant. They were anaesthetized in seawater with 4% magnesium chloride prior to coelomic fluid collection as shown in Fig.3.B. 10 krpm Insoluble fraction 50 Soluble Soluble fraction Fig.4. 1DE of the several fractions of the coelomic fluid obtained during centrifugation speed optimization. At 50 krpm it is possible to pull down several proteins and maintain different ones in the supernatant. 3 Searching for neuropeptides in the Soluble fraction of the Coelomic fluid 4 LC(RF C18)-Micro-ESI-MS LTQ, Thermofinningan MALDI-TOF MS Voyager, PerseptiveBiosystems 2DE of the Coelomic fluid Insoluble fraction 5 10 15 20 25 30 Time (min) 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 20,97 20,34 20,02 21,71 19,63 21,91 19,39 17,82 11,45 11,55 22,29 17,59 31,83 26,19 26,50 16,60 12,08 7,08 9,43 NL: 1,01E6 m/z 1000 5000 POROS R1 POROS R2 Gaphit 4527.3 2859.5 2837.5 4432.3 1169.7 1885.9 1522.0 2930.4 4510.1 1997.8 2994.8 1282.7 3050.6 3165.6 3786.7 2157.9 2270.7 3065.6 4322.7 4548.9 2583.9 4088.8 2060.9 1783.0 1818.7 1810.1 1506.2 1760.8 1153.4 1827.2 1447.4 1394.5 1266.3 1055.9 1129.6 976.75 1st Dimension 100 g of Insoluble fraction of the coelomic fluid was diluted in 8M Ureia, 2M tiureia, 2% CHAPS, 1% DTT, 2% IPG Buffer (Bouley, 2004, Proteomics, 4:1811-1824) until a final volume of 100 L. 1st dimension was performed in a IPGphor system (Amersham Biosystems), using immobiline SryStrip of 7cm wit a non-liner pH gradient from 3-10 (Amersham Biosystems). The isoelectric focosing program used was: 12H at 30V, 1H at 150V, 1H at 250V, 1H 1500V, 1H at 2500V, a voltage gradient of 14 until 8000V and 6H at 8000V. Fig.5. Micro-ESI MS Chromatogram of the Coelomic fluid soluble fraction. 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 740,92 635,25 888,75 756,67 940,17 688,33 806,08 590,25 537,33 1050,92 1127,83 524,17 1146,50 1265,33 1327,42 1506,75 700 800 900 1000 1100 1200 1300 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1013,25 950,08 1085,67 894,25 1169,00 844,67 1266,33 800,25 1212,67 1381,33 963,25 906,75 760,33 1058,08 856,33 1312,50 1112,83 724,00 (A) (B) 2nd Dimension Strips from IEF were equilibratred in a two-step process in 50mM Tris-HCl (pH 8.8) wit Ureia (6M), glycerol (30%, v/v), 2% SDS; w/v). DTT at 10mg/mL was used in the first equilibration and iodoacetamide at 40mg/mL in the second equilibration. Each equilibration was of 15 min under slow agitation. 2nd dimension was performed in SDS gels containing 12% acrylamide (w/v). Electrophoresis was run at 30 mA. Fig.8. MALDI-TOF MS spectra of peptides in the soluble fraction of the coelomic fluid. Several packing materials were tested in order to obtain most of the peptides from the soluble fraction (POROS R1, POROS R2 and graphit). Fig.6. mESI-MS Spectra of peptides (A) and intact protein (B) eluting from the reverse fase column (C18). Protein masses were deconvoluted using ProMass for Xcalibur (Thermo). Width: 1 Pairwise cutoff: 0.7 Consensus cutoff: 0 Consensus analysis-SPECLUST average std N min max POROS R1 POROS R2 Graphit 843.976 1 859.93 875.883 976.244 0.0426 2 976.201 976.286 1056.28 0.0512 1056.23 1056.33 1152.89 0.0629742 3 1152.8 1152.93 1169.23 0.04913 1169.16 1169.27 1169.26 1191.12 1282.16 0.0515112 1282.08 1282.2 1282.19 1331.88 0.0599 1331.82 1331.94 1446.83 0.0635 1446.77 1446.89 1521.83 0.0587205 1521.75 1521.88 1521.87 1783.4 0.0673 1783.34 1783.47 1885.35 0.0808538 1885.24 1885.42 1885.4 1996.18 0.0631 1996.12 1996.24 2060.21 2836.55 0.157092 2836.33 2836.7 2836.62 2858.61 0.123184 2858.44 2858.73 2858.66 2929.42 4432.1 0.204668 4431.81 4432.29 4432.19 4509.53 4527.16 0.175056 4526.91 4527.28 ITMS + c ESI d Full ms2 811,39@30,00 [ 210,00-2000,00] 200 400 600 800 1000 1200 1400 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1023,76 1080,61 799,05 785,07 914,48 272,16 385,30 844,42 656,41 988,29 757,30 456,39 585,38 1372,57 573,35 1174,67 369,20 1273,72 476,31 255,13 Fig.9. 2DE of the Insoluble fraction of the coelomic fluid. 7cm Immobiline DrySrpis was used in the 1st dimension. Fig.7. mESI-MS/MS spectra of one of the peptides eluting from the column (m/z 811.39). No identification was achieved on Bioworks (Thermo) using Uniprot database and a specific Equinoderm database (cDNA database). Table.1. Consensus analysis of the peptides found in the soluble fraction of the coelomic fluid using the three different packing materials. There are a total of 22 peptides rancking from m/z 840-4503. 2DE will be optimized to use 13 cm Immobiline DryStrips in the 1st dimension On going work in de novo sequencing peptides and proteins using several different proteases and de novo sequencing software PEAKS ©. Acknowledgments: Work supported by FCT throught a pHD grant to C. Franco (SFRH/BD/29799/2006)